The invention furthermore relates to an optical apparatus for microscopic investigation of a plurality of samples, having a sample holder that holds a plurality of samples and is supported movably, in particular in motorized and/or automatic fashion, relative to a sample illumination position in such a way that at least one of the samples is respectively successively positionable in the sample illumination position.
The need exists in some applications, for example for particular serial investigations or statistical investigations, to microscopically investigate a plurality of samples in succession, rapidly and preferably automatically. DE 199 50 225 A 1, for example, discloses a particular arrangement for allowing a large number of specific detection regions, which are distributed in grid fashion on a carrier of up to 22×60 mm, to be scanned with a confocal scanning microscope. According to the teaching of this document, the problem that the carrier is substantially larger than the object field of a usual scanning microscope is solved with the aid of an arm, pivotable in a plane parallel to the carrier, that carries some of the optical components.
Such a solution is not only very complex but also has the disadvantage that three-dimensional imaging of a plurality of samples would still take a very long time; this is not acceptable in particular with usually lifetime-limited, light-sensitive samples.
Many samples to be investigated microscopically have a limited lifetime. The reason for this circumstance is often, among others, their light-sensitivity. Three-dimensional scanning, for example using a scanning microscope, of samples arranged, for example, in the form of a matrix on a specimen slide is in this regard problematic in practice, since the scanning operation requires a great deal of time, and sample regions that lie outside the currently relevant scanning point are also respectively impinged upon by illumination light during the scanning operation and thereby damaged.
An entirely different technology, in which the sample is illuminated in layers, in principle allows more rapid acquisition of image data with less impact on samples, but hitherto has not been suitable for rapid mass investigations of samples, because the optical apparatuses required are technically very complex and because the samples must be separately and individually arranged in particular apparatuses and the observation apparatus must be laboriously configured individually for each sample. This method is known in particular as single plane illumination microscopy (SPIM).
A microscope operating with the SPIM method is described in DE 102 57 423 A1. With this microscope, a sample is illuminated with a thin light stripe while observation occurs perpendicularly to the plane of the illuminating light stripe. Illumination and detection occur here via two separate optical beam paths each having a separate optical system, in particular having two separate, mutually perpendicular objectives. The light stripe is generated by an illumination objective and a cylindrical optic preceding it. For image acquisition, the sample is moved through the light stripe that is stationary with respect to the detector, in order to acquire fluorescent light and/or scattered light in layers with a planar detector. The layer image data thereby obtained can then be assembled into a data set corresponding to a three-dimensional image of the sample.
In particular, this method as a rule cannot be implemented using apparatuses that have a conventional microscope structure with a standard microscope stand. Complex and expensive special arrangements are instead needed in order to implement these techniques.
DE 10 2004 034 957 A1 discloses an arrangement for microscopic observation of a sample via a microscope objective in whose housing light guides are provided, outside the lens optic, for the illumination light to the sample. The illumination light initially proceeds parallel to the optical axis of the objective inside the light guide, and then encounters an annular reflector of small aperture, attached to the objective housing, that focuses the illumination light with the aid of additional imaging elements into the sample, perpendicularly to the optical axis of the microscope objective and thus perpendicularly to the observation direction. Here as well, the sample is illuminated in area fashion using the SPIM principle. With this embodiment, positioning the sample inside the annular reflector is particularly problematic. An apparatus of this kind is therefore unsuitable for utilization of an automated method for serial investigation of a plurality of samples.
It is also known, especially for the investigation of aquatic organisms, not to hold the samples with a sample holder but instead to aspirate them through a glass capillary that is transilluminated with a light sheet. A procedure of this kind is known, for example, from the article by Burns et al., “Preparation strategy and illumination of three-dimensional cell cultures in light sheet-based fluorescence microscopy,” Journal of Biomedical Optics, Vol. 17(19), 101518 (October 2012). A procedure of this kind is unsuitable for structured, systematic, and targeted microscopic investigation of a plurality of samples. The article by Yanik et al., “Technologies for Micromanipulating, Imaging, and Phenotyping Small Invertebrates and Vertebrates,” Annu. Rev. Biomed. Eng. 2011; 13:185-217, also discloses a method in which the samples are pumped through a capillary.